This invention relates to a method for the in vitro amplification of closed circular DNA. More particularly, the invention relates to a process for the cell-free amplification of closed circular DNA for applications such as mutagenesis, molecular cloning, DNA detection, DNA modification, gene hunting, gene therapy, and cell-free DNA production.
The polymerase chain reaction (PCR) is a powerful method for the rapid and exponential amplification of target nucleic acids. E.g., U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated by reference. PCR has facilitated the development of gene characterization and molecular cloning technologies including direct sequencing of PCR-amplified DNA, the determination of allelic variation, and the detection of infectious and genetic disorders. PCR is performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing of opposing primers to the complementary DNA strands, and extension of the annealed primers with a DNA polymerase. Multiple PCR cycles result in the exponential amplification of the nucleotide sequence delineated by the flanking amplification primers.
An important modification of the original PCR technique was the substitution of Thermus aquaticus (Taq) DNA polymerase in place of the Klenow fragment of E. coil DNA polymerase I. Saiki et al., 230 Science 1350-1354 (1988). The incorporation of a thermostable DNA polymerase into the PCR protocol obviates the need for repeated enzyme additions and permits elevated annealing and primer extension temperatures, which enhance the specificity of primer/template associations. Other thermostable DNA polymerases have also been discovered and commercialized, such as the thermostable DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase; U.S. Pat. No. 5,545,552, hereby incorporated by reference), the thermostable DNA polymerase from Thermus flavus (Tfl DNA polymerase; Epicentre Technologies), the thermostable DNA polymerase from Thermus thermophilus (Tth DNA polymerase, Epicentre Technoloigies, Madison, Wis.), a mixture of Taq DNA polymerase and Pyrococcus species GB-D thermostable DNA polymerase (ELONGASE(trademark), Life Technologies, Inc., Gaithersburg, Md.), the thermostable DNA polymerase from Thermococcus litoralis (VentR(copyright) DNA polymerase, New England Biolabs, Beverly, Mass.), and AMPLITHERM(trademark) DNA polymerase (proprietary thermostable DNA polymerase, Epicentre Technologies). Thermostable DNA polymerases thus serve to increase the specificity and simplicity of PCR. PCR can be used to amplify linear DNA segments, but not closed circular DNA. Since most replicatively competent DNAs exist in closed circular form, for functional studies a PCR product needs to be subcloned into a closed circular DNA vector and then introduced into and amplified in appropriate cellular hosts. The ability to amplify circular DNA in vitro in a cell-free system would allow modification of the nucleotide composition and sequence of a circular DNA at will. This achievement would represent a significant advancement in the art and have potential applications in areas of mutagenesis, cloning, DNA detection, DNA modification, gene hunting, gene therapy, and cell-free DNA production.
It is an object of the present invention to provide a method of producing and amplifying closed circular DNA in vitro.
It is another object of the invention to provide a method of modifying the nucleotide composition and sequence of a closed circular DNA and amplifying the modified closed circular DNA in vitro.
It is still another object of the invention to provide a method of selectively amplifying a closed circular DNA having a defined nucleotide sequence in vitro.
These and other objects can be addressed by providing a method for in vitro amplification of a selected closed circular DNA comprising:
(a) mixing an effective amount of a thermostable DNA ligase with a PCR reaction mixture comprising the selected closed circular DNA as a template and a pair of 5xe2x80x2-phosphorylated amplification primers, with the proviso that any cofactor required for activity of the thermostable DNA ligase is also added in an effective amount, to result in an LDA mixture; and
(b) thermocycling the LDA mixture through a selected number of cycles of (i) a temperature suitable for denaturing the template, (ii) a temperature suitable for annealing the primers to the denatured template, (iii) a temperature suitable for polymerase-catalyzed extension of the primers, and (iv) a temperature suitable for ligase-catalyzed closing of the extended primers to result in an amplified closed circular DNA product.
In one illustrative embodiment of the invention, the temperature suitable for polymerase-catalyzed extension of the primers is the same as the temperature suitable for ligase-catalyzed closing of the extended primers. The temperature suitable for annealing the primers to the denatured template can also be the same as the temperature suitable for polymerase-catalyzed extension of the primers.
In preferred embodiments, the LDA mixture is held at the temperature suitable for denaturing the template for about 1 second to about 2 minutes in each cycle; the LDA mixture is held at the temperature suitable for annealing the primers to the denatured template for about 1 second to about 5 minutes in each cycle; and the LDA mixture is held at the temperature suitable for polymerase-catalyzed extension of the primers for about 1 to 20 minutes in each cycle.
When at least one of the primers has at least one mismatch with its corresponding template strand, the resulting amplified closed circular DNA product comprises a mutation as compared to the template. When at least one of the primers hybridizes to its corresponding template strand across a cloning site for an insert in a cloning vector, the resulting amplified closed circular DNA is in one orientation of the insert in the cloning vector. When the PCR reaction mixture farther comprises a modified nucleotide, the amplified closed circular DNA product comprises the modified nucleotide residue incorporated randomly therein. When at least one of the primers comprises a modified nucleotide, the amplified closed circular DNA product has the modified nucleotide residue incorporated at a pre-selected site.